The viral and cellular BCL-2 family of anti-apoptosis proteins exhibit three major activities: suppression of apoptosis, oncogenic cooperation and restraining of cell proliferation. The oncogenic activity of the anti-apoptosis proteins appears to be linked to the anti-apoptosis activity. To elucidate how these proteins suppress apoptosis and restrain cell proliferation, we will test two major hypotheses. (1) We will test the hypothesis that one of mechanisms by which the mammalian anti-apoptosis proteins promote cell survival may involve suppression of a protease (caspase) cascade initiated and amplified by a set of BCL-2 family pro-apoptotic protein such as BIK, BNIP1 and BNIP3. We postulate that BIK, BNIP1 and BNIP3 recruit and activate caspases involved in initiation of apoptosis in cytoplasmic locales where these pro-apoptotic proteins are expressed. Additionally, we suggest that BIK may also amplify protease activation after its own proteolytic processing. We will investigate if BIK, BNIP1 or BNIP3 physically associate with and activate various initiator caspases. Since mitochondrial dysfunction plays an important role in initiation of apoptosis, we will determine the effect of the mitochondrial pro-apoptotic protein BNIP3 on mitochondrial integrity and release of apoptotic factors from mitochondria. (2). The proliferation restraining activity of the anti-apoptosis proteins is separable from their anti-apoptosis activity. We hypothesize that these proteins modulate the activity of certain cell cycle regulatory proteins. We will determine if E1B-19K and EBV-BHRF1 proteins associated with and antagonize the activity of known cyclin/CDK complexes. We will also attempt to identify the protein kinases associated with the anti-apoptosis proteins by biochemical fractionation of HeLa cell extracts. The proposed studies should illuminate the mechanisms by which E1B-19K and related BCL-2 family proteins control cellular life and death cycles.